The present invention relates to a process for the production of a biologically active phenolic compound (+) catechin of formula 1 from Taxus wallichiana tissue cultures: 
In recent years, different Taxus species have attracted world wide attention due to the presence of taxol or its analogues in the bark or needles of the trees. Taxol, a highly oxygenated diterpenoid molecule and a potent anticancer drug was first isolated from the stem bark of Taxus brevifolia. Thereafter, it has also been isolated from other Taxus species including Taxus wallichiana. 
Catechins, the basic structural unit of condensed tannins, belong to flavan-3-ol derivatives and are found in a wide variety of plant sources such as vegetables, herbs and teas (Phytochem (1981) 20:869). Considerable interest has been expressed regarding the various pharmacological functions of catechins, which have been proved to be antibacterial, antiviral, anti-tumour, antioxidant and radical scavengers (Phytochem (1998) 49:2379-82).
The direct manipulation of plant cell and tissue culture systems has resulted in an enhanced production of various secondary metabolites. In vitro production of catechins, mainly (xe2x88x92) epicatechin-3-O-gallate accompanied by (+) catechin and (xe2x88x92) epicatechin have been reported in Fagophyrum esculentum calli and hairy root cultures (Phytochem (1992) 31:1239-1241, Phytochem (1993) 32:929 suspension cultures of Camellia sinensis (ChayeKexue (1995) 15:111-116) and Vitis vinifera (Biotech Lett. (1996) 18:659-662), Crataegus monogyna (Phytochem (1994) 37:1273), Uncaria elliptica (Phytochem (1998) 28:1099-1 100) and Polygonum hydropiper (Phytochem (1998) 49:1935-39).
Several approaches have been used for the establishment of in vitro cultures of various Taxus species (Plant Cell, Tissue and Organ Cult (1996) 46:59-75). Different explants and various basal media have been used for the initiation and maintenance of Taxus callus and cell suspension cultures. The culture media are frequently supplemented with organic substances, such as casein hydrolysate, polyvinylpyrrolidone, ascorbic acid and others. Several growth regulators are used for stimulation of cell proliferation. Taxus wallichiana, known as Himalayan yew is available in India. Suspension and callus cultures of Taxus wallichiana are found capable of producing taxol (Planta Med. (1998) 64:270-72) and some important taxanes, namely 2-deacetoxy-taxinine J and 2-deacetoxy austrospicatin (Planta Med. (1996) 62:333-35). The Applicants have been screening different callus lines of Taxus wallichiana induced from different explants of trees collected from different geographical regions of India. The protocol standardized for in vitro callus production is dependent on media composition (viz. Murashige and Skoog""s, Gambrog""s, White""s, Nitsch and Nitsch""s), hormonal regime combinations of different concentrations of cytokinins and auxins, (such as 6-benzyl aminopurine, TDZ, 2-ip, Kinetin, 6-methylamino purine, Zeatin with NAA, IAA, IBA, 2,4-D, 2,4-T, Picloram), explant source (preferably from needles, twigs, stems devoid of needles and seeds) and culture conditions (light or dark conditions).
The callus line developed from a specific explant on different media compositions having definite hormonal combinations resulted in the production of a phenolic compound. The compound, having a molecular formula of C15H14O6, mp 94-95xc2x0 C., was isolated as an amorphous solid. The compound has been characterized as (+) catechin. The yield was noted to be 0.3% in six months old callus. The (+) catechin is very important precursor for the synthesis of other catechin derivatives, eg. Gallocatechin, epigallocatechin and epigallocatechin-3-O-gallate. It is worth mentioning here that most of the earlier reports of in vitro catechin production revealed production of a mixture of catechins, the majority of which were (xe2x88x92) epicatechin, (xe2x88x92) eipcatechin-3-O-gallate and (xe2x88x92) epigallocatechin, (Phytochem (1989) 28:1099-1100, (1992) 31:1239-1241 (1993) 32:924-931, (1998) 49:1935-1939), whereas in the present invention only (+) catechin is expressed. Furthermore, in the present invention 0.3% concentration of (+) catechin has been detected in 6 months old callus while the expression of (+) catechin in Polygonum hydropiper occurred after one year of culture initiation (Phytochem (1998) 49(7):1935-39).
Thus the main object of the present invention is to provide a process for In vitro production of a biologically active phenolic compound (+) catechin of formula I from Taxus wallichiana tissue cultures.
Another object of the invention is to provide a process for isolation of (+) catechin from cell cultures of Taxus wallichiana without the use of any cumbersome chromatographic separations.
The present invention consisting of the in vitro production of (+) catechin from Taxus wallichiana constitute the first ever report of production of (+) catechin in the genus Taxus. Moreover, the yield of (+) catechin obtained in the invention is 0.3% which is significantly better to the so far reported ones from cell culture sources taking into consideration of the reported culture period (Phytochemistry (1998) 49(7):1935-1939).
Accordingly the present invention relates to a process for the production of a biologically active phenolic compound (+) catechin of formula I from Taxus wallichiana tissue cultures which comprises: (a) inoculation of explants on culture media supplemented with combinations of auxins (1-5 mg/l) and cytokinins (0.1-1.0 mg/l); (b) incubation of the cultures under continuous light or dark conditions for 4-6 weeks for callus initiation followed by subculturing at 4-6 weeks intervals; (c) extraction of fresh pulverized calli with polar solvents at room temperature, (d) evaporating the polar solvents to obtain a residue, and (e) treatment of the residue with a chlorinated solvent and isolation of the compound (+) catechin by filtration.
In an embodiment, the explants for induction of callus may be selected from needles, twigs, stems devoid of needles and seeds of Taxus Wallichiana. 
In another embodiment, the culture media for callus induction and multiplication are selected from Murashige and Skoog, (1962) (MS) medium, containing the following (in mg/l)xe2x80x94NH4NO3 (1,650), KNO3 (1,900), CaCl2.2H2O (400), MgSO4.7H2O (370), KH2PO4 (170), Na2EDTA.2H2O (7.2), FeSO4.7H2O (27.8), MnSO4.4H2O (22.3), ZnSO4.7H2O (8.6), H3BO3 (6.2), KI (0.83), Na2MoO4.2H2O (0.25), CuSO4.5H2O (0.025), CoCl2.6H2O (0.025), Glycine (2.0), Nicotinic acid (0.5), PyridoxineHCl (0.5), ThiamineHCl (0.1); Gamborg""s; (1968) (B5) medium, containing the following (in mg/l)xe2x80x94KNO3 (3,000), (NH4)2SO4 (134), MgSO4.7H2O (500), CaCl2.2H2O (150), NaH2PO4.H2O (150), MnSO4.H2O (10.0), KI (0.75), H3BO3 (3.0), ZnSO4.7H2O (2.0), CUSO4 (0.025), NaMoO4.2H2O (0.25), CoCl2.6H2O (0.025), Na2EDTA.2H2O (37.2) FeSO4.7H2O (27.8), White""s (1963) medium, consisting of the following (in mg/l) xe2x80x94Ca(NO3)2 (142.0), KNO3 (81.0), MgSO4.7H2O (70.0), KCl (65.0), KH2PO4 (12.0), (FeSO4)3 (2.46) and Nitsch and Nitsch; (1969) medium, containing the following (in mg/l)xe2x80x94NE4NO3 (20.0), KNO3 (950), H3BO3 (10.0), KH2PO4 (68.0), Na2MoO4.2H2O (0.143), CaCl2 (41.5), MgSO4.7H2O (185), MNSO4.4H2O (15.0), ZnSO4.H20 (10.0), CuSO4 (0.14), FeSO4 (111.4), Na2EDTA (149), Biotin (0.05), Glycine (2.0), Nicotinic acid (5.0), Pyridoxine HCl (0.5), Thiamine HCl (0.5), Folic acid (5.0).
In yet another embodiment, the auxins may be selected from Indole acetic acid (IAA), naphthalene acetic acid (NAA), indole butyric acid (IBA), 2,4-dichlorophenoxy acetic acid (2,4-D), 2,4,6,-trichlorophenoxy acetic acid (2,4-T) and picloram within the following range (1.0-5.0 mg/l).
In still another embodiment, the cytokinins may be selected from 6-benzyl amino purine (BAP), 6-methyl aminopurine (MAP), kinetin (Kn), zeatin, thiadiazuron (TDZ) and 2-isopentenyl amino purine (2-ip) within a range of about 0.1-1 mg/l.
In still another embodiment, the cultures are incubated under continuous light of 300-3000 lux or under continuous dark conditions.
In still another embodiment, harvesting time to get maximum product may be from one month to thirty six months
In still another embodiment, the polar solvents may be selected from methanol, ethanol, propanol and butanol.
In still another embodiment, the ratio of auxin and cytokinin used ranges between 6 to 20:1.
In still another embodiment, the medium may be supplemented with casein hydrolysate in an amount ranging between 100-400 mg/l.
In still another embodiment, ascorbic acid used ranges between 10-50 mg/l.
In still another embodiment, of the present invention the chlorinated solvent may be selected from chloroform and dichloromethane.
Repeated experimentations have proved that use of particular explants and specific ratio of auxins and cytokinins are the critical factors for in vitro expression of (+) catechin production.
The invention is described in detail in the examples given below which are provided to illustrate the invention and therefore should not be construed to limit the scope of the invention.